Dimeric compounds and their use as anti-viral agents

ABSTRACT

The invention relates to compounds of general formula (I); in which R is an amino or guanidino group; R 2  is acetyl or trifluoroacetyl; X is CONH, SO 2 NH, NHCO or NHCONH; m is either 0 or 1; n is an integer from 2 to 6; q is an integer from 0 to 3; and Y is hydrogen or an aromatic substituent, or a pharmaceutically acceptable derivative thereof; methods for their preparation, pharmaceutical formulations containing them or their use in the prevention or treatment of a viral infection

This invention relates to new chemical compounds and their use inmedicine. In particular the invention concerns novel dimeric compounds,methods for their preparation, pharmaceutical formulations thereof andtheir use as anti-viral agents.

BACKGROUND OF THE INVENTION

Enzymes with the ability to cleave N-acetyl neuraminic acid (NANA), alsoknown as sialic acid, from other carbohydrates are present in manymicroorganisms. These include bacteria such as Vibrio cholerae,Clostridium perfringens, Streptococcus pneumoniae and Arthrobactersialophilus, and viruses such as influenza virus, parainfluenza virus,mumps virus, Newcastle disease virus and Sendai virus. Most of theseviruses are of the orthomyxovirus or paramyxovirus groups, and carry aneuramimidase activity on the surface of the virus particles. Many ofthese neuramimidase-possessing organisms are major pathogens of manand/or animals, and some, such as influenza virus and Newcastle diseasevirus, cause diseases of enormous importance.

It has long been thought that inhibitors of neuramimidase might preventinfection by neuramimidase-bearing viruses. Most of the knownneuramimidase inhibitors are analogues of neuraminic acid, such as2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) and some of itsderivatives (Meindl et al, Virology, 1974 58 457). Our InternationalPatent Publication No. WO 91/16320 describes a number of analogues ofDANA which are active against viral neuramimidase, and it has been shownin particular that 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminicacid (Compound (A), code number GG167) is useful in the treatment ofinfluenza A and B (N. Engl. J. Med., 1997 337 874–880). Other patentapplications describe various closely-related sialic acid derivatives(eg. PCT Publications No. WO 95/18800, No. WO 95/20583 and No. WO98/06712), and anti-viral macromolecular conjugates of GG167 have alsobeen described (International Patent Application No. PCT/AU97/00771).

International Patent Publication No. WO 00/55149, describes dimericcompounds which comprise two neuramimidase binding molecules, such ascompound (A), attached to a common spacer or linking group of up to 100atoms in length.

We have now discovered a novel class of compounds which fall within thegeneric scope of International Patent Publication No. WO 00/55149, butwhich are not specifically disclosed therein, and exhibit a surprisinglyadvantageous anti-influenza activity profile which includes a long lungresidency time and high potency.

Without wishing to be bound by theory, the basis for the long residencytime in the lungs is thought to be due to the size and molecular weightof the compounds preventing entry through tight junctions in therespiratory epithelium and the polarity of the compounds being such thatpassage through the cell membranes occurs very inefficiently. Analternative theory is that the compounds themselves interact with thephospholipids in the cell membrane or other components of therespiratory epithelium and increase the residency time in the lungs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides for a compound ofgeneral formula (I):

in which

R is an amino or guanidino group;

R² is acetyl or trifluoroacetyl;

X is CONH, SO₂NH, NHCO or NHCONH;

m is either 0 or 1;

n is an integer from 2 to 6;

q is an integer from 0 to 3; and

Y is hydrogen or an aromatic substituent,

or a pharmaceutically acceptable derivative thereof.

Preferably R is a guanidino group.

Preferably R² is an acetyl group.

Suitable aromatic substituents include alkyl, alkenyl, alkynyl, aryl,halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy,alkenyloxy, aryloxy, carboxy, benzyloxy, haloalkoxy, haloalkenyloxy,haloaryloxy, nitro, azido, amino, alkylamino, alkenylamino,alkynylamino, arylamino, benzylamino, acylamino, acyl, arylacyl,acylamino, acyloxy, alkylsulphonyl, arylsulphonyl, sulphonylamino,alkylsulphonylamino, arylsulphonylamino, heterocyclyl, heterocycloxy,heterocyclylamino, haloheterocyclyl, mercapto, sulfonic acid, alkylthio,arylthio and acylthio.

Preferably the alkyl, alkenyl, alkynyl and alkoxy substituents containup to 6 carbon atoms.

More preferably Y is hydrogen, halogen, C₁₋₄ alkyl, hydroxy, C₁₋₄alkoxy, amino or carboxy.

It will be appreciated by those skilled in the art that the compounds offormula (I) may be modified to provide pharmaceutically acceptablederivatives thereof at any one or more of the functional groups in thecompounds of formula (I). Of particular interest as such derivatives arecompounds modified at the carboxyl function, hydroxyl functions or atamino groups. Thus compounds of interest include alkyl esters, such asmethyl, ethyl, propyl or isopropyl esters, aryl esters, such as phenyl,benzoyl esters, and acetyl esters of the compounds of formula (I).

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ether, ester or salt of such ester ofa compound of formula (I) or any other compound which, uponadministration to the recipient, is capable of providing a compound offormula (I) or an anti-virally active metabolite or residue thereof. Ofparticular interest as derivatives are compounds modified at the sialicacid carboxy or glycerol hydroxy groups, or at amino and guanidinegroups.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicylic, succinic,toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic,benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonic acids.Other acids such as oxalic acid, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (eg. sodium),alkaline earth metal (eg. magnesium), ammonium, and NR₄ ⁺ (where R isC₁₋₄alkyl) salts.

The compounds of the invention may be prepared by methods describedherein. It will be apparent to those skilled in the art, that it isnecessary to use protecting groups to protect one or more functionalgroups of the neuramimidase binding molecule during the process ofattaching the monomers to the alkyl spacer group. See for example“Protective Groups in Organic Synthesis” by T. W. Green and P. G. M.Nuts (John Wiley & Sons, 1991). Pharmaceutically acceptable salts of thecompounds of formula (I) may be prepared according to known procedures.

For ease of processing and preparation, it is preferable that thecompounds of formula (I) are in crystalline form.

Accordingly, the present invention also provides a method for thepreparation of the compound of formula (I) as defined above, whichcomprises the step of deprotecting a compound of formula (II)

in which R and R² are as defined above and P₁ is a carboxylic acidprotecting group.

The compounds of formula (I) possess antiviral activity. In particularthese compounds are inhibitors of viral neuramimidase oforthomyxoviruses and paramyxoviruses, for example the viralneuramimidase of influenza A and B, parainfluenza, mumps and Newcastledisease.

Thus in a second aspect the invention provides a compound of formula (I)or a pharmaceutically acceptable derivative thereof, for use as anactive therapeutic agent in the treatment of a viral infection, forexample orthomyxovirus and paramyxovirus infections.

In a third aspect the invention provides a method for the prevention ortreatment of a viral infection comprising the step of administration toa subject in need thereof of an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt or derivativethereof.

Preferably, the viral infection is an orthomyxovirus or paramyxovirusinfection. More preferably the viral infection is an influenza A or Binfection.

Preferably the subject is an animal such as a mammal, more preferably ahuman, or a member of the genus Equus, for example a horse, donkey ormule. Most preferably the mammal is a human.

In a fourth aspect the invention provides use of a compound of theinvention for the manufacture of a medicament for the treatment of aviral infection.

As used herein, the term “effective amount” is meant an amount of thecompound of formula I effective to preventing or treating a viralinfection in order to yield a desired therapeutic response. For example,to overcome or alleviate the effects of a viral infection.

The term “therapeutically-effective amount” means an amount of thecompound of formula I to yield a desired therapeutic response. Forexample, treating or preventing a viral infection.

The specific “therapeutically-effective amount” will, obviously, varywith such factors as the particular viral infection being treated, thephysical condition of the subject, the type of animal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulation employed and the structure of the compoundor its derivatives.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a viral infection or signor symptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a viral infection. “Treating” as used herein covers anytreatment of, or prevention of a viral infection in a vertebrate, amammal, particularly a human, and includes: (a) preventing the viralinfection from occurring in a subject that may be predisposed to theviral infection, but has not yet been diagnosed to the viral infection,but has not yet been diagnosed as having it; (b) inhibiting the viralinfection, ie., arresting its development; or (c) relieving orameliorating the effects, i.e., cause regression of the symptoms of theviral infection.

The compounds of the invention may also be used in diagnostic methods,in particular methods for the detection of influenza virus. For use insuch methods it may be advantageous to link a compound of the inventionto a label, such as a radioactive, fluorescent or chemiluminescentlabel.

Methods of diagnosis for which the compounds of the invention aresuitable are described, for example, in our earlier applicationsPCT/AU97/00109 and PCT/AU97/00771.

In a fifth aspect the invention provides a method for the detection of aviral infection which comprises the step of contacting the compound ofthe invention with a sample suspected of containing the virus.

It will be further appreciated that the amount of a compound of theinvention required for use in treatment will vary not only with theparticular compound selected but also with the route of administration,the nature of the condition being treated, and the age and condition ofthe patient, and will ultimately be at the discretion of the attendantphysician or veterinarian. In general however, a suitable dose will bein the range of from about 0.001 to 100 mg/kg of bodyweight per day,preferably in the range of 0.01 to 10 mg/kg/day, most preferably in therange of 0.1 to 1 mg/kg/day.

Treatment is preferably commenced before or at the time of infection andcontinued until virus is no longer present in the respiratory tract.However the compounds are also effective when given post-infection, forexample after the appearance of established symptoms.

Suitably treatment is given on one or two occasions, preferably onlyonce only for treatment, and preferably once per week for prophylaxis.

The compound is conveniently administered in unit dosage form, forexample containing 1 to 100 mg, more conveniently 1 to 20 mg of activeingredient per unit dosage form.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical, it is preferable topresent the active ingredient as a pharmaceutical formulation.

Thus in a sixth aspect the invention provides a pharmaceuticalformulation comprising a compound of formula (I) or a pharmaceuticallyacceptable salt or derivative thereof, together with one or morepharmaceutically acceptable carriers therefor and, optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not being deleterious to the recipient thereof.

The compounds of the invention may also be used in combination withother therapeutic and/or prophylactic agents, for example otheranti-infective agents. In particular the compounds of the invention maybe employed with other antiviral agents. The invention thus provides ina seventh aspect a combination comprising a compound of formula (I) or apharmaceutically acceptable salt or derivative thereof together withanother therapeutically and/or prophylactically active agent, inparticular an antiviral agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus such formulationscomprising a combination as defined above together with apharmaceutically acceptable carrier therefor comprise a further aspectof the invention.

Suitable therapeutic and/or prophylactic agents for use in suchcombinations include other anti-infective agents, in particularanti-bacterial and anti-viral agents such as those used to treatrespiratory infections. For example, other compounds or vaccineseffective against influenza viruses, such as the sialic acid analoguesreferred to above, e.g. zanamivir, oseltamivir, amantadine, rimantadineand ribavirin and FluVax, may be included in such combinations.

The individual components of such combinations may be administeredeither separately, sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When the compounds of the invention are used with a second therapeuticand/or prophylactic agent active against the same virus, the dose ofeach compound may either be the same as or different from that employedwhen each compound is used alone. Appropriate doses will be readilyappreciated by those skilled in the art.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administration,or those in a form suitable for administration to the respiratory tract(including the nasal passages) for example by inhalation orinsufflation. The formulations may, where appropriate, be convenientlypresented in discrete dosage units, and may be prepared by any of themethods well known in the art of pharmacy. These methods include thestep of bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may for examplebe in the form of aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles, which may includeedible oils, or preservatives.

The compounds according to the invention may also be formulated forparenteral administration by injection, for example bolus injection, orcontinuous infusion, and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulating agents such as suspending, stabilising and/ordispersing agents. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilisation from solution, for constitution with a suitable vehicle,eg. sterile, pyrogen-free water, before use.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase, and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and gum acacia or gum tragacanth; pastilles comprising theactive ingredient in an inert base such as gelatin or sucrose and gumacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping moulds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For administration to the respiratory tract, including intranasaladministration, the neuramimidase inhibitors may be administered by anyof the methods and formulations employed in the art for administrationto the respiratory tract.

Thus in general the compounds may be administered in the form of asolution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for exampleprepared from water alone (for example sterile or pyrogen-free water) orwater and a physiologically acceptable co-solvent (for example ethanol,propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipientsfor example preservatives (such as benzalkonium chloride), solubilisingagents/surfactants such as polysorbates (eg. Tween 80, Span 80,benzalkonium chloride), buffering agents, isotonicity-adjusting agents(for example sodium chloride), absorption enhancers and viscosityenhancers. Suspensions may additionally contain suspending agents (forexample microcrystalline cellulose, carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase a means of dose metering is desirably provided. In the case of adropper or pipette this may be achieved by the patient administering anappropriate, predetermined volume of the solution or suspension. In thecase of a spray this may be achieved for example by means of a meteringatomising spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the compound is provided in apressurised pack with a suitable propellant, such as achlorofluorocarbon (CFC), for example dichlorodifluoromethane,trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of drug may be controlled byprovision of a metered valve.

Alternatively the compounds may be provided in the form of a dry powder,for example a powder mix of the compound in a suitable powder base suchas lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). Conveniently the powdercarrier will form a gel in the nasal cavity. The powder composition maybe presented in unit dose form, for example in capsules or cartridges ofeg. gelatin, or blister packs from which the powder may be administeredby means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size, for example of the order of 5 microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronisation.

When desired, formulations adapted to give sustained release of theactive ingredient may be employed.

Preferably the compounds of the invention are administered to therespiratory tract by inhalation, insufflation or intranasaladministration, or a combination thereof.

“Relenza” is administered by oral inhalation as a free-flow powder via a“Diskhaler” (Oseltamivir). A similar formulation would be suitable forthe present invention.

Thus, according to an eighth aspect of the present invention there isprovided an inhaler which contains a formulation as defined above.

It will be appreciated that the inhaler may also be in the form of ameter dose aerosol inhaler.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

TABLE 1

Com- Aryl pound Substituentl Aryl Group No Y substitution m Group XGroup n 1 H 1,3 0 CONH 4 2 H 1,3 1 NHCO 2 3 H 1,3 0 SO₂NH 4 4 H 1,4 1NHCO 2 5 H 1,4 0 CONH 3 6 H 1,4 0 CONH 6 7 H 1,2 0 CONH 6 8 H 1,3 0 CONH6 9 5-CO₂H 1,3 0 CONH 3 10 5-CH₃ 1,3 0 NHCONH 6 11 H 1,3 0 CONH 3 12 H1,2 0 CONH 3 13 H 1,4 1 CONH 3 14 H 1,2 1 CONH 3Machine MethodsGreen Method (LC/MS)

Micromass Platform II mass spectrometer operating in positive ionelectrospray mode, mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min    Purple Method (Mass Directed Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm)

-   UV wavelength: 200–320 nM-   Flow: 20 ml/min-   Injection Volume: 1 ml-   Solvent A: 0.1% formic acid-   Solvent B: 95% acetonitrile+5% formic acid-   Gradient: 100% A/1 min, 100–80% A/9 min, 80–1% A/3.5 min, 1% A/1.4    min, 1–100% A/0.1 min    Turquoise Method (Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm).

-   UV wavelength: 230 nm-   Flow: 4 ml/min-   Injection Volume: 2 ml-   Solvent A: acetonitrile+0.05% TFA-   Solvent B: water+0.1% TFA    Method A (LC/MS)

Micromass Platform II mass spectrometer operating in positive ionelectrospray mode, mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min    Method B (LC/MS)

Waters ZQ mass spectrometer operating in positive ion electrospray mode,mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min,    Method C (Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm).

-   UV wavelength: 230 nm-   Flow: 4 ml/min-   Injection Volume: 2 ml-   Solvent A: acetonitrile+0.05% TFA-   Solvent B: water+0.1% TFA-   Gradient: 0–40% A/20 min, 40% A/20 min, 40–100% A/0.3 min, 100% A/15    min, 100–0% A/3 min    Method D (Mass Directed Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm)

-   UV wavelength: 200–320 nM-   Flow: 20 ml/min-   Injection Volume: 1 ml-   Solvent A: 0.1% formic acid-   Solvent B: 95% acetonitrile+5% formic acid-   Gradient: 100% A/1 min, 100–80% A/9 min, 80–1% A/3.5 min, 1% A/1.4    min, 1–100% A/0.1 min    Abbreviations-   EtOAc ethyl acetate-   MeOH methanol-   HPLC high pressure liquid chromatography-   SPE solid phase extraction-   LC/MS Liquid chromatography/mass spectroscopy-   DMF N,N-Dimethylformamide-   WSCDI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide-   HOBt 1-hydroxybenzotriazole-   DIPEA N,N-diisopropylethylamine-   MeCN acetonitrile-   RT room temperature-   EtOAc ethyl acetate-   MgSO₄ magnesium sulphate-   DMF dimethylformamide

EXAMPLE 1 Preparation of Compound 1

(4S, 5R,6R)-5-Acetylamino-4-azido-6-[(S)-4-nitrophenoxycarbonyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)methyl]-5,6-dihydro-4H-pyran-2-carboxylicacid methylester (see Eur. J. Med. Chem. 1999, 34, 563–574) (2.00 g, 3.8mmol) was dried by azeotroping 3 times from anhydrous toluene thendissolved in anhydrous acetonitrile (20 ml) with the addition of a few 3angstrom molecular sieve pellets. The stirred solution was treated withN-tert-butoxycarbonyl 1,4-diaminobutane (0.72 g, 3.8 mmol) andtriethylamine (0.43 g, 4.2 mmol). The mixture was stirred for 16 h undera nitrogen atmosphere. Volatiles were removed in vacuo to afford ayellow residue. This was redissolved in EtOAc (50 ml), washed with0.5MHCl (30 ml) then brine (30 ml). The solution was dried (Na₂SO₄) andsolvent evaporated in vacuo to afford a cream coloured foam. Furtherpurification was by Biotage flash chromatography, eluant initiallyEtOAc:Cyclohexane (1:1) then EtOAc. Evaporation of solvent in vacuo gaveIntermediate 1 (1.26 g, 58% yield) as a white solid. LC/MS (Method B)showed MH⁺=571; T_(RET)=2.87 min

Intermediate 1 (0.76 g, 1.33 mmol) was dissolved in ethanol (24 ml) andsubjected to catalytic hydrogenation over Lindlar Catalyst (0.095 g) for16 h. Catalyst was removed by filtration and evaporation of solvent invacuo gave Intermediate 2 (0.72 g, 99% yield) as a cream coloured foam.LC/MS (Method A) showed MH⁺=545; T_(RET)=2.24 min

Intermediate 2 (0.72 g, 1.32 mmol) was dissolved in tetrahydrofuran (7ml) and treated with N,N′-bis-(tert-butoxycarbonyl)-1-guanylpyrazole(0.45 g, 1.45 mmol). The mixture was stirred under a nitrogen atmospherefor 16 h. Volatiles were removed in vacuo to give a solid residue whichwas purified by Biotage flash chromatography; eluant initiallyEtOAc:Cyclohexane (1:1) then EtOAc:Cyclohexane (5:3). Evaporation ofsolvent in vacuo afforded Intermediate 3 (0.48 g, 46% yield) as a whitesolid. LC/MS (Method A) showed MH⁺=787; T_(RET)=3.64 min

Intermediate 3 (0.48 g, 0.61 mmol) was dissolved in dichloromethane (19ml). The solution was cooled in an ice bath and trifluoroacetic acid (19ml) was added portionwise over 5 minutes. The mixture was then stirredfor 1 h under a nitrogen atmosphere before being allowed to warm toambient temperature and stirred a further 16 h. Volatiles were removedin vacuo, and the residue azeotroped from toluene to remove remainingtrifluoroacetic acid. Trituration with diethyl ether (20 ml) afforded awhite solid which was separated to give Intermediate 4 (0.50 g). LC/MS(Method B) showed (M−H)⁻=485; T_(RET)=0.52 min.

Intermediate 4 (0.050 g, 0.07 mmol) was dried by azeotroping 3 timesfrom anhydrous toluene and then dissolved in a mixture of anhydrousacetonitrile (2 ml) and anhydrous DMF (1 ml). The solution was treatedsuccessively with isophthalic acid (0.005 g, 0.03 mmol), HOBt (0.010 g,0.07 mmol), WSCDI (0.013 g, 0.07 mmol) and DIPEA (0.026 g, 0.20 mmol).The mixture was stirred for 16 h under a nitrogen atmosphere. Volatileswere removed in vacuo to afford a yellow residue which was purified bymass-directed preparative HPLC (method D) to give Intermediate 5 (0.013g) as a white solid. LC/MS (Method A) showed (M+2H⁺)/2=552; T_(RET)=2.05min

Intermediate 5 (0.012 g, 0.01 mmol) was dissolved in a mixture of water(0.5 ml) and methanol (0.5 ml). The resulting solution was treated withtriethylamine (0.090 g, an excess). The mixture was stirred for 50 minbefore rapid removal of volatiles in vacuo to give a white solidresidue. Purification by preparative HPLC (method C) gave Compound 1(0.002 g) as a white solid. LC/MS (Method B) showed (M+2H⁺)/2=512;T_(RET)=1.77 min

EXAMPLE 2 Preparation of Compound 2

(4S, 5R,6R)-5-Acetylamino-4-azido-6-[(S)-4-nitrophenoxycarbonyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)methyl]-5,6-dihydro-4H-pyran-2-carboxylicacid methylester (see Eur. J. Med. Chem. 1999, 34, 563–574) (4.0 g) wasazeotroped with toluene (50 mL) and dissolved in MeCN (40 mL) andtriethylamine (1.12 mL) and 3-aminopropionic acid t-butyl esterhydrochloride (1.396 g) added. After 3 days at RT, the solvent wasremoved and the residue diluted with EtOAc (150 mL). This was washedwith 5% citric acid solution (2×50 mL), dried (MgSO₄) and concentrated.Purification by Biotage eluting with 1:1 cyclohexane:EtOAc, then 60:40then 65:35 cyclohexane:EtOAcgave Intermediate 6 as a colourless foam(3.45 g).

¹H-NMR (400 MHz, CDCl₃) δ 6.72 (d, 1H), 5.97 (d, 1H), 5.53 (t, 1H), 5.40(t, 1H), 5.03–4.95 (m, 2H), 4.92 (dd, 1H), 4.74–4.64 (m, 2H), 3.83 (s,3H), 3.64–3.54 (m, 1H), 3.38–3.27 (m, 2H), 2.65–2.56 (m, 1H), 2.52–2.43(m, 1H), 2.06 (s, 3H), 1.70 (s, 1H), 1.48 (s, 9H).

Similarly prepared to Intermediate 2 from Intermediate 6. LC/MS (greenmethod) MH⁺ 504, T_(RET)=2.22 min

Intermediate 8 was prepared similarly to Intermediate 3 fromIntermediate 7

LC/MS (green method) MH⁺ 744, T_(RET)=3.66 min

Intermdiate 8 (1.44 g), trifluoroacetic acid (20 mL), dichloromethane(20 mL) and anisole (2 mL) were stirred at RT for 3 h after which thevolatiles were removed in vacuo. The residue was triturated with Et₂O(2×25 mL) and then dried in vacuo to afford Intermediate 9 as a whitesolid (1.22 g).

LC/MS (green method) MH⁺ 488, T_(RET)=1.25 min

Intermediate 9 (0.12 g), DIPEA (0.14 mL), m-xylylenediamine (0.0132 mL)and benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate(0.104 g) were mixed at RT for 2 days and then concentrated in vacuo.Purification using mass directed HPLC (purple method) gave Intermediate10 (0.038 g).

LC/MS (green method) MH⁺/2 538, T_(RET)=1.74 min

Intermediate 10 (0.038 g), water (1.5 mL), methanol (1.5 mL) andtriethylamine (0.5 mL) were mixed at RT for 1 h. The volatiles wereremoved in vacuo and the aqueous residue acidified to pH4 withtrifluoroacetic acid. Reverse phase HPLC (turquoise method) eluting with0–17.5% MeCN over 30 min afforded Compound 2 (7.6 mg).

LC/MS (green method) MH⁺/2 497, T_(RET)=1.64 min

EXAMPLE 3 Preparation of Compound 3

Intermediate 4 (0.075 g, 0.11 mmol) was dried by azeotroping 3 timesfrom anhydrous toluene. The dried solid was dissolved in a mixture ofanhydrous chloroform (2 ml) and anhydrous DMF (1 ml) with addition ofDIPEA (0.056 g, 0.43 mmol). The resulting clear solution was added to1,3-Benzenedisulphonyl chloride (0.014 g, 0.05 mmol) and the mixturestirred for 1 h under a nitrogen atmosphere. Volatiles were removed invacuo to give a residue which was further purified by reverse phase SPE(eluant 15% aqueous acetonitrile) to afford Intermediate 11 (0.020 g) asa white solid. LC/MS (Method B) showed (M+2H⁺)/2=588; T_(RET)=2.09 min

Intermediate 11 (0.020 g, 0.014 mmol) was dissolved in a mixture ofwater (0.5 ml) and methanol (0.5 ml). The resulting solution was treatedwith triethylamine (0.090 g, an excess). The mixture was stirred for 50min before rapid removal of volatiles in vacuo to give a white solidresidue. Purification by preparative HPLC (method C) gave Compound 3(0.006 g) as a white solid. LC/MS (Method A) showed (M+2H⁺)/2=548;T_(RET)=1.99 min.

EXAMPLE 4 Preparation of Compound 4

Similarly prepared to Intermediate 10 from Intermediate 9 andp-xylylenediamine to afford Intermediate 12 (0.038 g). LC/MS (greenmethod) MH⁺/2 538, T_(RET)=1.94 min

Compound 4 was similarly prepared to Compound 2 from Intermediate 12.

LC/MS (green method) MH⁺/2 497, T_(RET)=1.62 min

EXAMPLE 5 Preparation of Compound 5 by Reaction of Intermediate 13 andTerephthalic Acid

The aminopropyl Intermediate 13 was prepared following a similarsequence of steps to that described for the analogous aminobutylIntermediate 4 in Example 1.

Terephthalic acid (5.93 mg, 0.0357 mmole), Intermediate 13 (50 mg, 0.071mmole) and benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) (37.7 mg, 0.0852 mmole) were dissolved in DMF(2 ml) to which was added di-isopropylethylamine (DIPEA, 91.8 mg, 0.71mmole). The resulting mixture was stirred at room temperature for 5hours. The reaction mixture was purified by reverse phase HPLC using aWaters Symmetry C18 column (5 micron 19×100 mm), and gradient elution asshown in the following Table, to afford the protected dimer (19.5 mg,51%), MS 1075 (M+H)⁺

Time Flow rate (minutes) A % B % (ml/min) 0 100 0 6 2 100 0 6 22 40 60 632 40 60 6 35 100 0 6 42 100 0 6 A = Water containing 0.1%trifluoroacetic acid B = Acetonitrile containing 0.06% trifluoroaceticacid

The protected dimeric compound (19.5 mg, 0.0182 mmole) was dissolved ina mixture of water/methanol/triethylamine in the ratio 4:4:1 (2 ml) andstirred at room temperature for 1 hour then evaporated to dryness underreduced pressure. Remaining triethylamine was removed by repeatedaddition of water and evaporation under reduced pressure. The remainingresidue was purifed by reverse phase HPLC using a Waters Symmetry C18column (5 micron, 19×100 mm), and gradient elution as shown in thefollowing Table, to afford the dimer 5 as a white solid (10.8 mg, 60%)after freeze-drying.

Time Flow rate (minutes) A % B % (ml/min) 0 100 0 6 2 100 0 6 22 60 40 632 60 40 6 35 100 0 6 42 100 0 6 A = Water containing 0.1%trifluoroacetic acid B = Acetonitrile containing 0.06% trifluoroaceticacid

MS 498.3 (M+2H)²⁺, 995.2 (M+H)⁺

¹H-nmr (D₂O) δ (ppm): 1.79 (t, 4H); 1.90 (s, 6H); 3.14 (t, 4H); 3.42 (m,6H); 3.62 (dd, 2H); 3.98 (m, 2H); 4.08 (dd, 2H); 4.37 (dd, 2H); 4.52(dd, 2H); 4.88 (dd, 0.2H); 5.92 (d, 2H); 7.79 (s, 4H).

EXAMPLE 6 Preparation of Compound 5 by Reaction of Intermediates 14 and15

Intermediate 14

To a suspension of terephthalic acid (141.9 mg, 0.85 mmole) andN-Boc-1,3-diaminopropane (300 mg, 1.72 mmole) in DMF (12 ml) was addedtriethylamine (392 mg, 3.88 mmole) with stirring at room temperature. Tothe white suspension was added in portions BOP (771 mg, 1.74 mmole).After addition of the first portion of BOP (about 300 mg) the reactionmixture became a clear solution. The reaction mixture was stirred atroom temperature for 16 hours and then evaporated in vacuo to removeDMF. The residue was stirred in water (10 ml) at room temperature for 3hours and then the solid collected by filtration. The solid was washedwith water (4×5 ml) and filtered. The filter cake was dissolved in hotmethanol (4 ml) and then diluted with water (9 ml). The suspension wasstirred at room temperature for 3 hours and then filtered. The solid waswashed with water (4×5 ml), then dissolved in boiling methanol (4 ml)and cooled to room temperature. The crystalline suspension was allowedto agitate at room temperature overnight. The solid was filtered off,washed with cold methanol and air-dried to give the bis-amido product asa white solid (365 mg, 89.7%), MS 479 (M+H)⁺ The Boc-protected bis-amide(360 mg) was stirred in a mixture of trifluoroacetic acid (TFA) (2 ml)and dichloromethane (2 ml) at room temperature for 4 hr, then evaporatedto dryness under vacuum. The residue was dissolved in water (6 ml) andfreeze-dried to remove any excess TFA and afford the Intermediate 14(TFA salt) as a white solid (379 mg, 98%), MS 279 (Base +H)+

Intermediate 15

A solution of methyl (2R, 3R, 4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate(113 mg, 0.197 mmol) in dry pyridine (3 ml) containing4-dimethylaminopyridine (120 mg, 0.982 mmol) was treated with4-nitrophenylchloroformate (199 mg, 0.987 mmol) at 22° C. The mixturewas stirred at 22° C. for 17 h, then the pyridine removed in vacuo. Theresidue was purified by SPE chromatography (5 g cartridge) eluting withcyclohexane-ethyl acetate (4:1–2:1) to afford Intermediate 15 as a paleyellow gum (96 mg, 66%).

NMR δ(CDCl₃) 11.3 (1Hs, NH), 8.58 (1H brd, NH), 8.26 (2H, AA′BB′,aromatic CH's), 7.56 (2H, AA′BB′, aromatic CH's), 6.82 (1H brd, NH),5.93 (1Hd, ═CH), 5.54 (1Hdd, CH), 5.20 (1Hdt, CH), 5.10 (1Hdt, Ch), 4.78(2Hm, 2×CH), 4.44 (1H brq, CH), 4.28 (1Hdd, CH), 3.82 (3Hs CH3), 1.91(3Hs, CH3), and 1.48 (18Hs, 2× tert butyl).

LCMS R_(t)=3.87 min. (MH⁺=738, MH⁻=736)

Compound 5

Intermediate 14 (38.9 mg, 0.0768 mmole) and Intermediate 15 (2equivalents) were dissolved in pyridine (0.5 ml) and the resultingmixture was stirred at room temperature for 16 hr and then evaporated todryness under vacuum. The residue was partitioned betweendichloromethane (15 ml) and water (3 ml). The organic layer was washedwith water (2×2 ml), then evaporated to dryness to give a gummy residuewhich was chromatographed on silica gel (20 g, solvent ethylacetate/methanol 10/1). The protected form of Compound 5 was thusisolated as a white foam (56 mg, 49%).

The protected compound (50 mg, 0.0339 mmole) was treated with TFA andthen aqueous triethylamine as described in Example 5 to give Compound 5(16.8 mg, 50%), MS 995 (M+H)⁺.

EXAMPLE 7

Compounds 6 to 14 (Table 1) of the invention were prepared in ananalogous manner to the Examples given above and the NMR and/or massspectral data is given below.

Compound 6

MS 540.6 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm); 1.43 (br, 8H); 1.51 (br, 4H); 1.65 (br, 4H); 2.00(s, 6H); 3.12 (m, 4H); 3.41 (m, 4H); 3.43 (dd, 2H); 3.70 (dd, 2H); 4.07(dd, 2H); 4.18 (dd, 2H); 4.46 (dd, 2H); 4.58 (dd, 2H); 4.98 (dd, 2H);5.98 (d, 2H); 7.85 (s, 4H).

Compound 7

MS 1079 (M+2H)²⁺, 540.6 (M+2H)²⁺, 360.5 (M+3H)³⁺

Compound 8

MS 1079.5 (M+2H)²⁺, 540.25 (M+2H)²⁺, 360.5 (M+3H)³⁺

¹H-nmr (CD₃OD) δ (ppm): 1.35 (br, 8H); 1.45 (br, 4H); 1.55 (br, 4H);1.90 (s, 6H); 3.03 (m, 4H); 3.35 (m, 4H); 3.44 (dd, 2H); 3.56 (dd, 2H);3.95 (m, 2H); 4.15 (dd, 2H); 4.35 (dd, 2H); 4.52 (dd, 2H); 4.90 (dd,2H); 5.82 (d, 2H); 7.48 (t, 1H); 7.88 (d, 2H); 8.19 (s, 1H).

Compound 9

MS 1039.1 (M+H)⁺, 520.3 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm): 1.79 (t, 4H); 1.90 (s, 6H); 3.19 (t, 4H); 3.46 (m,6H); 3.60 (dd, 2H); 4.00 (m, 2H); 4.08 (dd, 2H); 4.37 (dd, 2H); 4.47(dd, 2H); 4.88 (dd, 2H); 5.88 (d, 2H); 8.28 (s, 1H); 8.49 (s, 2H).

Compound 10

MS 1123.7 (M+H)⁺, 562.3 (M+2H)²⁺

¹H-nmr (CD₃OD) δ (ppm): 1.40 (br, 8H); 1.51 (br, 8H); 1.93 (s, 6H); 2.19(s, 3H); 3.08 (br, 4H); 3.19 (br, 4H); 3.52 (dd, 2H); 3.65 (dd, 2H);4.01 (m, 2H); 4.16 (dd, 2H); 4.35 (dd, 2H); 4.53 (dd, 2H); 4.98 (dd,2H); 5.89 (d, 2H); 7.10 (dd, 2H); 7.40 (s, 1H).

Compound 11

MS 995.1 (M+H)⁺, 498.3 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm): 1.75 (t, 4H); 1.90 (s, 6H); 3.15 (t, 4H); 3.45 (m,6H); 3.60 (dd, 2H); 3.98 (m, 2H); 4.05 (dd, 2H); 4.35 (dd, 2H); 4.45(dd, 2H); 4.92 (dd, 2H); 5.91 (d, 2H); 7.50 (t, 1H); 7.92 (d, 2H); 8.07(s, 1H).

Compound 12

MS 995.3 (M+H)⁺, 498.3 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm): 1.78 (t, 4H); 1.92 (s, 6H); 3.15 (t, 4H); 3.40 (t,4H); 3.46 (dd, 2H); 3.60 (dd, 2H); 3.95 (m, 2H); 4.00 (dd, 2H); 4.35(dd, 2H); 4.50 (dd, 2H); 4.93 (dd, 2H); 5.83 (d, 2H); 7.62 (dd, 4H).

Compound 13

MS 1023.1 (M+H)⁺, 512.3 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm): 1.65 (t, 4H); 1.85 (s, 6H); 3.02 (t, 4H); 3.15 (t,4H); 3.42 (dd, 6H); 3.52 (s, 4H); 3.59 (dd, 2H); 3.98 (m, 2H); 4.12 (dd,2H); 4.40 (dd, 2H); 4.50 (dd, 2H); 4.90 (dd, 2H); 5.90 (d, 2H); 7.30 (s,4H).

Compound 14

MS 1023.3 (M+H)⁺, 512.4 (M+2H)²⁺

¹H-nmr (D₂O) δ (ppm): 1.61 (t, 4H); 1.91 (B, 6H); 3.00 (t, 4H); 3.13 (t,4H); 3.40 (dd, 6H); 3.57 (s, 4H); 3.58 (dd, 2H); 3.99 (m, 2H); 4.10 (dd,2H); 4.40 (dd, 2H); 4.50 (dd, 2H); 4.90 (dd, 2H); 5.88 (d, 2H); 7.32 (m,4H).

EXAMPLE 8 Large Scale Preparation and Purification of Compound 5

TFA Salt to HCl Salt

The 2TFA salt (log) of Compound 5 prepared by Example 6 was dissolved inMeOH/water (1:1 v/v) (150 ml) and applied to a prewashed AmberliteIRA-410 (chloride form) ion exchange column (4×50 cm). The 2HCl salt ofCompound 5 was eluted from this column with MeOH/water (1:1 v/v).Fractions containing the 2HCl salt were concentrated in vacuo to give awhite foam.

Deprotection

The 2HCl salt (10.4 g) was dissolved in methanol (144 ml) and water (144ml) and cooled in ice. Triethylamine (7.75 ml) was added in threeportions over 10 minutes. The reaction was monitored by reverse phaseHPLC. When the reaction was complete, the reaction mixture wasevaporated to dryness in vacuo.

A solution of impure Compound 5 containing triethylamine was evaporatedto dryness to yield a solid (10.5 g). This solid was dissolved in 80 mlof water and 5 ml of orthophosphoric acid was added to ensure the samplesolution was pH 2. This solution (ca 100 ml) was subjected topreparative HPLC using a 7 micron Kromasil C8 column (25 cm×5 cm id.).Chromatographic resolution of Compound 5 was achieved using ion-pairgradient elution where the start mobile phase A was H₂O to which hadbeen added 8 g/liter of sodium lauryl sulphate (SLS) and 2 ml/literH₃PO₄. The final mobile phase B was 60% CH₃CN/H₂O to which had beenadded 8 g/liter SLS and 2 ml/liter H₃PO₄. Gradient time was 0 to 100% Bin 70 minutes holding at 100% B for 20 minutes. Flow was set at 80ml/min. A total of 4 injections at 25 ml each were done. The detectorwas set at 260 nm. The fractions, 50 ml each, were bulked according totheir analytical purity as measured by HPLC, the specificationbeing >97.5%. This bulked volume was approximately 1800 ml for 4chromatographic separations. To remove the majority of the phosphate andthe lauryl sulphate ions, the aqueous bulk was added to 1200 ml of IRA410 ion exchange resin in the chloride form and the solution stirred for1 hour. The resin was removed by filtration and washed with 400 ml ofH₂O which was combined with the filtrate. The aqueous phase was thenpassed through a column of IRA 410 in the chloride form (15 cm×2.5 cmid.) to remove any remaining phosphate or lauryl sulphate ioncontamination. The column was then washed with 50 ml H₂O and combinedwith the filtrate. After removal of the CH₃CN by rotary evaporation at40° C., the pH was adjusted to 7.0. The aqueous phase was split into 2portions. The first portion was passed through a column of Amberchrom CG161 (25 cm×2.5 cm) in order to adsorb Compound 5. This column is apolystyrene divinyl benzene resin which acts as a reversed phase packingmaterial. The column was washed with 300 ml of H₂O until the wash wasclear of chloride ion, this being checked by the absence of a reactionwith AgNO₃ solution. The column was eluted with 30% CH₃CN/H₂O (500 ml).This process was repeated with the second portion and the eluentscombined. The solvents (1 liter) were removed by rotary evaporation toyield 5.7 g of solid.

The purified material was taken up in water (300 ml) and washed withdichloromethane (3×200 ml). The aqueous solution was then freeze-driedto give the title compound.

¹H NMR (D₂O) δ 7.85 (s, 4H, 4× ArH), 5.67, 5.57 (2×d, 2H, J=2.1 Hz,2×CH), 4.95 (dd, 2H, J=9.0, 1.6 Hz, 2×CH), 4.52 (dd, 2H, J=10.5, 1.6 Hz,2×CH), 4.42 (dd, 2H, J=9.3, 2.2 Hz, 2×CH), 4.14–4.04 (m, 4H, 4×CH), 3.67(dd; 2H, J=12, 3 Hz, 2×CH), 3.54–3.44 (m, 6H, 2×CH+2×CH₂), 3.21 (t, 4H,J=6.7 Hz, 2×CH₂), 1.96 (s, 6H, 2×CH₃), 1.85 (m, 4H, 2×CH₂).

Microanalysis

Calc: C, 45.02%; H, 6.23%; N, 15.75%. Found: C, 44.80%; H, 6.68%; N,14.89%.

LC-MS (Method A) showed (M+2H)²⁺=498; T_(RET)=1.67 min

EXAMPLE 9 Evaluation of the Compounds of Formula (I)—Inhibition ofInfluenza Virus Replication

Cytopathic effect (CPE) assays were performed essentially as describedby Watanabe et al. (J. Virological Methods, 1994 48 257). MDCK cellswere infected with a defined inoculum of virus (determined byexperimentation to be the minimum sufficient to cause adequate CPE in 72hours and to be susceptible to control compounds at concentrationsconsidered to be consistent with published norms) in the presence serialdilutions of Compounds of the invention. Cultures were incubated for upto 72 hours at 37° C. in a 5% CO₂ atmosphere. The extent of CPE andhence viral replication was determined via metabolism of the viral dye3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)according to published methods (see for example, Watanabe et al., 1994).The compound concentration that inhibited CPE by 50% (ID₅₀) wascalculated using a computer program for curve fitting. InfluenzaA/Sydney/5/97 and B/Harbin/7/95 viruses were assayed and the results areshown in Table 2. Comparable data for a specifically disclosed compoundin WO 00/55149 and for compound A is also shown in Table 2.

TABLE 2 ID₅₀ μg/ml ID₅₀ μg/ml Description A/Sydney/5/97+ B/Harbin/7/95Compound A 0.023 +/− 0.024 0.013 +/− 0.011 Compound 2 0.0002 0.0004Compound 3 0.0001 0.00007 Compound 4 0.0003 0.0004 Compound 5 0.00020.0007 Compound 6 0.0002 0.0009 Compound 7 0.0007 0.0008 Compound 80.0004 0.0009 Compound 9 0.0001 0.0003 Compound 10 0.0002 0.0006Compound 11 0.0001 0.0001 Compound 12 >0.1 0.0001 Compound 13 0.00010.0003 Compound 14 0.0002 0.0003 Compound Number 8* 0.0007, 0.0005 0.007+/− 0.01  Compound Number 10* 0.057 >0.1 *As referenced in WO 00/55149+Data provided in WO 00/55149 related to the virus H3N2 isolateA/Victoria/3/75 rather than A H3N2 isolate A/Sydney/5/97. When comparingsuch data the person skilled in the art will appreciate that differencesin antiviral potency are not uncommon for a given compound when analysedagainst several different viruses in vitro. For example, Woods etal(Antimicrob Agents Chemother 1993 37: 1473–9) have reported thatCompound A exhibits a wide range of EC50 values (from 0.02 to 0.16 uM)in in vitro assays involving recent clinical isolates. Accordingly,compound 8 was found to be more potent in CPE assays involving therecent influenza A H3N2 isolate A/Sydney/5/97 than the earlier H3N2isolate A/Victoria/3/75.

Data provided in Table 2 demonstrate that the compounds 2–14, inaddition to being substantially more potent than the highly activecompound A, are even more potent against A/Sydney/5/97 and substantiallymore potent against the recent influenza B isolate B/Harbin/7/95 thancompounds 8 and 10 of WO 00/55149.

EXAMPLE 10 Plaque Reduction Assay

Madin Darby Canine Kidney (MDCK) cells are seeded into six well tissueculture plates and grown to confluency via standard methods. Influenzaviruses are diluted in a minimal volume of phosphate buffered salinesupplemented with 0.2% bovine serum albumin to yield an estimated titreof 50–100 plaque forming units (pfu) per well. After adsorption to theMDCK cells for one hour at 37° C. in a 5% CO₂ atmosphere the viralinocula is aspirated and replaced with viral growth media (minimalEagle's media supplemented with BSA, trypsin andinsulin/transferrin/selenium at optimal concentrations) containingsufficient agar or agarose (generally 1–2%) to cause the media to gel atroom temperature and at 37° C. in a 5% CO₂ atmosphere until plaquesdevelop (generally 2–4 days). Plaques can be visualised with a suitablestain (e.g. 0.4% crystal violet in formal saline) before counting.Antiviral potency is expressed as the concentration of test articlewhich reduces plaque numbers by 50% of the untreated control value(EC₅₀).

EC₅₀ ng/ml PRA Example A/WSN* A/Vic* A/Syd* A/New* A/Pan* A/Bay*Compound 56, >100 5.5 +/− 2.4 0.27, 2.7, 3 35 A 8.2 0.23 5 0.02 0.0030.05 0.06 0.03 0.009 Amantadine 220 11 157 Oseltamivir 0.11 0.23 0.3*A/WSN/33 BVLV09 (H1N1) A/Victoria/3/75 BVLV017 (H3N2) A/Sydney/5/97BVLV015 (H3N2) A/New Caledonia/20/99 BVLV008 (H1N1) A/Panama/2007/99BVLV008 (H3N2) A/Bayern/7/95 BVL006 (H1N1)

EC₅₀ ng/ml PRA Example B/HongK* B/Yam* A 21 +/− 6 0.2, 3.1 5 0.316 0.022Amantadine >10000 2061 Tamlflu 32 0.7 *B/Victoria/1/67 B/Hong Kong/5/72BVLV012 B/Harbin/7/95 BVLV008 B/Yamanashi/166/98 BVLV007

EXAMPLE 11 Assessment of Long Duration of Action

Rodents are anaesthetised and dosed with compound of interest by theintra-tracheal route at a dose volume of 0.8 ml/kg. The rodent is thenheld in the vertical position until full recovery is achieved. Atdifferent time points, for example, 2, 8, 24 and 48 hours post-dose,levels of compound in the lung tissue are assessed by analyticalmethods. Any analytical method suitable for detection of this type ofcompound may be used. The time at which levels of compound fall belowthe sensitivity of the analytical techniques identified will determinethe residency time of the compound in lung tissue.

The rat lung retention data for selected compounds is shown below.Please note that all experiments included a co-dosed internal standard,namely compound 3 of International Patent Publication No. WO 02/20514,to permit comparison. The data are expressed as a ratio with respect tothis compound, the structure of which is shown below.

The data for compound A is included for comparison purposes. Thecompounds of the invention have significantly greater retention at 7days than Compound A when expressed as a ratio of compound concentrationto standard concentration.

Rat lung retention assay results [PCT AU01/01128 Mean [PCT AU01/01128Ratio Mean [lung] time point dose [cmpd] Mean [cmpd] compound 3]compound 3] [cmpd]/PCT hrs Compound mg/kg ng/g ng/g ng/g ng/g AU01/01128compound 3 48 Compound 5 0.1 1325 1888 48 Compound 5 0.1 750 951 11381403 0.68 48 Compound 5 0.1 778 1184 168 Compound 5 0.1 636 1479 168Compound 5 0.1 300 403 761 984 0.41 168 Compound 5 0.1 272 712 48Compound 1 0.1 2281 4802 48 Compound 1 0.1 650 1278 1406 2874 0.44 48Compound 1 0.1 902 2413 168 Compound 1 0.1 280 629 168 Compound 1 0.1611 372 1568 915 0.41 168 Compound 1 0.1 227 549 48 Compound 3 0.1 24574802 48 Compound 3 0.1 675 1410 1406 2874 0.49 48 Compound 3 0.1 10972413 168 Compound 3 0.1 245 629 168 Compound 3 0.1 544 329 1568 915 0.36168 Compound 3 0.1 199 549 48 Compound A (zanamivir) 0.1 421 698 48Compound A (zanamivir) 0.1 369 352 1901 1368 0.26 48 Compound A(zanamivir) 0.1 267 1507 168 Compound A (zanamivir) 0.1 91 815 168Compound A (zanamivir) 0.1 47 61 925 750 0.08 168 Compound A (zanamivir)0.1 45 512

EXAMPLE 12 Alternative Assessment of Long Duration of Action andEfficacy

The protocol for infecting mice has been described previously (1–4).Mildly anaesthetised mice are inoculated into the external nares withinfluenza virus. Treatment procedure and regimen. A single dose ofcompound is administered at a defined time point up to 10 days prior toinfection, preferably 4–7 days prior to infection, or followinginfection, preferably immediately following infection and up to 48 hourspost infection. In most experiments, a non-lethal strain of influenza isused, and efficacy is assessed by reductions in lung virus titre. Formice given compound prior to infection, lungs are removed post infectioneither on a single day, or on days following infection, preferably days1–4 post infection. Homogenised lung samples are assayed for virus usingestablished methods, and the titres of viral load estimated and comparedto titres of virus in lungs of untreated mice.

In those experiments where a mouse-adapted lethal strain of influenza isused, efficacy is assessed by an increase in survival rate and/ornumbers of survivors, as compared to untreated mice.

REFERENCES

1. Ryan, D. M., J. Ticehurst, M. H. Dempsey, and C. R. Penn, 1994.Inhibition of influenza virus replication in mice by GG167(4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) isconsistent with extracellular activity of viral neuramimidase(sialidase). Antimicrob. Agents and Chemother. 38 (10): 2270–2275.

2. von Itzstein M., W.-Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B.Jin, T. V. Phan, M. L. Smythe, H. F. White, S. W. Oliver, P. M. Colman,J. N. Varghese, D. M. Ryan, J. M. Woods, R. C. Bethell, V. J. Hogham, J.M. Cameron, and C. R. Penn. 1993. Rational design of potentsialidase-based inhibitors of influenza virus replication. Nature(London) 363:418–423.

3. Woods, J. M., R. C. Bethell, J. A. V. Coates, N. Healey, S. A.Hiscox, B. A. Pearson, D. M. Ryan, J. Ticehurst, J. Tilling, S. A.Walcott, and C. R. Penn. 1993.4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highlyeffective inhibitor both of the sialidase (neuramimidase) and of growthof a wide range of influenza A and B viruses in vitro. Antimicrob.Agents Chemother. 37:1473–1479.

4. Robert J Fenton, Peter J Morley, Ian J Owens, David Gower, SimonParry, Lee Crossman and Tony Wong (1999). Chemoprophylaxis of influenzaA virus infections, with single doses of zanamivir, demonstrates thatzanamivir is cleared slowly from the respiratory tract. Antimicrob.Agents and Chemother. 43, 11, 2642–2647.

1. A compound of general formula (I)

in which R is an amino or guanidino group; R² is acetyl ortrifluoroacetyl; X is CONH, SO₂NH, NHCO or NHCONH; m is either 0 or 1; nis an integer from 2 to 6; q is an integer from 0 to 3; and Y ishydrogen or an aromatic substituent, or a pharmaceutically acceptablesalt, ether, ester or salt of such ester thereof.
 2. A compoundaccording to claim 1, in which R is a guanidino group.
 3. A compoundaccording to claim 1, in which R² is an acetyl group.
 4. A compoundaccording to claim 1, in which Y is hydrogen, halogen, C₁₋₄ alkyl,hydroxy, C₁₋₄ alkoxy, amino or carboxy.
 5. A compound according to claim1, which contains a pharmaceutically acceptable salt, ether, ester orsalt of such ester at one or more of the carboxyl groups, hydroxylgroups, amino groups or guanidine groups.
 6. A compound according toclaim 1, in which said compound is an alkyl ester, an aryl ester or anacetyl ester.
 7. A method for the preparation of the compound of formula(I) according to claim 1, which comprises the step of deprotecting acompound of formula (II)

in which R and R² are as defined in claim 1 and P₁ is a carboxylic acidprotecting group.
 8. A pharmaceutical formulation comprising atherapeutically effective amount of a compound of formula (I) as definedin claim 1 or a pharmaceutically acceptable salt, ether, ester or saltof such ester thereof, together with one or more pharmaceuticallyacceptable carriers.
 9. A pharmaceutical formulation according to claim8, which further comprises one or more anti-viral agents used to treatrespiratory infections.
 10. A pharmaceutical formulation according toclaim 9, in which the agent is zanamivir, oseltamivir, amantadine,rimantadine, ribavirin and/or FluVax.
 11. An inhaler which comprises acompound according to claim
 1. 12. An inhaler according to claim 11which is adapted for oral administration as a free-flow powder.
 13. Aninhaler according to claim 11 which is a metered dose aerosol inhaler.14. A method for the treatment of an orthomyxovirus or paramyxovirusinfection, comprising the step of administration to a subject in needthereof of an effective amount of a compound of formula (I) as definedin claim
 1. 15. A method according to claim 14 in which theorthomyxovirus or paramyxovirus infection is an influenza A or Binfection, parainfluenza, mumps or Newcastle disease.
 16. A methodaccording to claim 14 in which the administration is to the respiratorytract by inhalation, insufflation or intranasally or a combinationthereof.
 17. A method for the preparation of the compound of formula (I)according to claim 1, which comprises the steps of: (a) reacting acompound of formula (III):

in which R₂ is as defined in claim 1, L is a leaving group, P₁ is acarboxylic acid protecting group and P₂ is an amine protecting group,with a compound of the compound of formula (IV):H₂N(CH₂)_(n)NHP₂,  (IV) in which n is defined in claim 1, to form thecompound of formula (V):

in which n is defined in claim 1, P₁ is a carboxylic acid protectinggroup and P₂ is an amine protecting group, (b) deprotecting the compoundof formula (V) to form the compound of formula (VI):

(c) reacting the compound of (VI) with a compound of formula (VII) or(VIII):

in which m, Y, and q are as defined in claim 1, to form the compound offormula (II):

in which R and R² are as defined in claim 1 and P₁ is a carboxylic acidprotecting group, and (d) deprotecting the compound of formula (II). 18.A method for the preparation of the compound of formula (I) according toclaim 1, which comprises the steps of: (a) reacting a compound offormula (III):

in which L is a leaving group, P₁ is a carboxylic acid protecting groupand P₂ is an amine protecting group, with a compound of the compound offormula (IX):H₂N(CH₂)_(n)CO₂P₁,  (IX) in which n is defined in claim 1, to form thecompound of formula (X):

in which n is defined in claim 1, P₁ is a carboxylic acid protectinggroup and P₂ is an amine protecting group, (b) deprotecting a compoundof formula (X) to form a compound of formula (XI):

(c) reacting the compound of formula (XI) with a compound of formula(XII):

in which m, Y, and q are defined in claim 1, to form the compound offormula (II):

in which R and R² are as defined in claim 1 and P₁ is a carboxylic acidprotecting group, and (d) deprotecting the compound of formula (II). 19.A method for the detection of an orthomyxovirus or paramyxovirusinfection which comprises the step of contacting the compound of formula(I) as defined in claim 1 with a sample suspected of containing thevirus.
 20. A method according to claim 14, further comprising the stepof administering an effective amount of one or more anti-viral agentsused to treat respiratory infections.